Peptide complexes containing phospholipase d

ABSTRACT

The present invention provides peptide complexes comprising phospholipase D and one or morephospholipase D-interacting peptides. The peptide complexes are useful in screening assays foridentifying compounds effective in modulating the peptide complexes and in treating and/or preventing diseases associated with phospholipase D and it&#39;s interacting partners. In addition, methods for screening modulators of the peptide complexes or interacting members thereof are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationfrom PCT/KR03/001903, filed Sep. 18, 2003, designating the U.S. whichclaims benefit of U.S. Provisional Application 60/411,600 filed on Sep.18, 2002 and U.S. Provisional Application 60/416,552 filed on Oct. 8,2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an isolated peptide complex, morespecifically, to an isolated peptide complex comprising phospholipase D,and a screening method for modulators thereof.

2. Background Art Description of the Related Art

Mammalian phospholipase D (PLD) is an enzyme that hydrolyzesphosphatidyl choline (PC) into phosphatidic acid (PA) and choline inresponse to a variety of signals including hormones, neurotransmitters,and growth factors. PA is known as intracellular lipid secondmessengers, which are involved in multiple physiological events such aspromotion of mitogenesis, stimulation of respiratory bursts, secretoryprocesses, and actin cytoskeletal reorganization in many cells (Lee, S.,et al., J. Biol. Chem., 277:6542-6549 (2002)). PA can be also convertedinto various other signaling molecules such as diacylglycerol (DAG),lysophosphatidic acid (LPA), and arachidonate.

There are two mammalian PLD isoforms, PLD1 and PLD2. PLD2 is ˜50%identical to PLD1. Unless otherwise specified, the term “PLD” usedherein encompasses both PLD1 and PLD2. The essential feature thatdefines mammalian PLD is the presence of two catalytic domains. Inaddition, both mammalian PLDs contain a putative pleckstrin homology(PH) domain, a phox homology (PX) domain, and other conserved regions ofunknown function. PLD1 contains a “loop” sequence between two catalyticdomains, which is not found in PLD2 (Rizzo, M. A., et al., Pharmacol.Therapeutics, 94:35-50 (2002)). PLD activity in mammalian cells isregulated by various factors including proteins and lipids, for example,protein kinase C (PKC), small GTPases, and phosphatidylinositol4,5-bisphosphate (PIP2) (Melendez, A. et al., Immunology, 14:49-55(2002)). This indicates that PLD regulation is a complicated and tightlyregulated process. The ubiquitous nature of agonist-dependent PLDactivation suggests that PLD is involved in the regulation of importantcellular processes and it may function as a hub molecule in cellularsignaling (Exton J H, Biochim. Biophys. Acta, 1439, 121-133 (1997)).

Further, PLD is involved in budding, intracellular vesicle traffickingand vacuolar molecule sorting, formation of multivesicular bodies,endocytosis, tumorigenesis, cell transformation, and proliferation(Foster D A, Mol Cell Biol 21:595-602 (2001), Morris A J, BiochimBiophys Acta, 1439:175-86 (1999)) and it is thus associated withdiseases and disorders such as neurodegenerative diseases, autoimmunediseases, cancer, and diabetes (Min D S, J Biol Chem. 277:12334-42(2002), Farese R V, Am J Physiol Endocrinol Metab., 283:E1-11 (2002)).

Therefore, it is highly valuable to find a novel PLD binding partnerand/or a peptide complex of PLD and the PLD binding partner. That is, itcan be presumed that a peptide complex of PLD and a PLD binding partnermediates the functions of PLD and the binding partners in the biologicalprocesses or disease pathways. Therefore, a peptide complex comprisingPLD and a PLD binding partner will be useful for modulating biologicalprocesses or treating diseases by PLD (Smith S O, J Biol Chem., 278:21459-66. (2003)). Further, a peptide complex comprising PLD and a PLDbinding partner will be useful for screening a modulator of aninteraction between PLD and a PLD binding partner, which may be alsoused to modulate biological processes or to treat diseases with PLD. Asused herein, modulating an interaction between PLD and a PLD bindingpartner means altering (enhancing or reducing) the activities of PLDand/or a PLD binding partner, e.g., increasing the concentrations of PLDand/or a PLD binding partner, enhancing or reducing their biologicalactivities, increasing or decreasing their stability, altering theiraffinity or specificity to certain other biological molecules, etc.

For example, a modulator of the peptide complex, including agonist andantagonist, would be useful in treating diseases and disorders such asneurodegenerative diseases, autoimmune diseases, cancer and diabetes.

Therefore, undoubtedly, there is a continued need to develop a peptidecomplex comprising PLD and a PLD binding partner.

SUMMARY OF THE INVENTION

The present invention provides novel PLD binding partners, with whichPLD forms a peptide complex useful for modulating biological processesor treating diseases by PLD and/or the PLD binding partners and a methodfor screening modulators of the peptide complex.

In one aspect of the present invention, there is provided an isolatedpeptide complex comprising a first peptide selected from the groupconsisting of (a1) phospholipase D (PLD), (a2) a PLD variant, (a3) a PLDfragment, and (a4) a fusion peptide containing (a1), (a2), or (a3); anda second peptide selected from the group consisting of (b1) actin,aldolase, collapsin response mediator molecule-2 (CRMP-2), phospholipaseCγ-1 (PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1,glucose transporter 4 (GLUT4), mammalian target of rapamycin (mTOR),heat shock protein 70 (hsp7o), dynamin, munc 18, tubulin, n-nitric oxidesynthase (nNOS), integrin beta 3, guanine nucleotide exchange factor—H1(GEF-H1), V-ATPase, phosphoinositide-3-phosphate (PIP3), or dopaminetransporter (DAT), (b2) a variant of (b1), (b3) a fragment of (b1), and(b4) a fusion peptide containing (b1), (b2), or (b3). The first peptidemay be linked to the second peptide by a covalent bond.

In another aspect of the present invention, there is provided ascreening method for modulators of the peptide complex, which comprisesproviding the isolated peptide complex; contacting the isolated peptidecomplex with a test compound; and detecting an interaction between thetest compound and the isolated peptide complex and/or an interactionchange between the first peptide and the second peptide.

In still another aspect of the present invention, there is provided ascreening method for modulators of an interaction between the firstpeptide and the second peptide, which comprises contacting the firstpeptide with the second peptide in the presence of a test compound; anddetecting an interaction between the first peptide and the secondpeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1 shows detection of a 43-kDa protein as a PLD2-binding protein;

FIG. 2 shows interaction between actin and PLD2;

FIG. 3A shows the isolation of PLD2-binding protein (p40) by the aboveblot overlay assay;

FIG. 3B shows the matrix-assisted laser desorption ionization massspectrum of the digested peptides of p40;

FIG. 4 shows direct interaction between aldolase and PLD2;

FIG. 5 shows detection of a p62 as a PLD2-binding protein;

FIG. 6 shows direct interaction between CRMP-2 and PLD2-PX;

FIG. 7 shows direct interaction between PLC-γ1 and PLD2;

FIG. 8 shows detection of a p35 as a PLD2-binding protein;

FIG. 9 shows direct interaction between Akt and PLD2;

FIG. 10 shows association with PLD1 and GLUT4;

FIG. 11 shows a direct binding of purified PLD1 to the cytoplasmiccentral loop of GLUT4;

FIGS. 12 and 13 show detection of HSP70 as a PLD2-binding protein;

FIG. 14 shows direct interaction between HSP70 and PLD2;

FIGS. 15A and 15B show detection of dynamin as a PLD-binding protein;

FIG. 16 shows direct interaction between dynamin and PLD2;

FIG. 17 shows detection of munc-18-1 as a PLD -binding protein;

FIG. 18 shows direct interaction between munc-18-1 and PLD2;

FIG. 19 shows detection of p55 protein as a PLD2-binding protein;

FIG. 20 shows that the 55-kDa protein is co-precipitated with PLD2 inCOS-7 cells is α- and β-tubulin;

FIG. 21 shows detection of nNOS as a PLD2-binding protein;

FIG. 22 shows interaction between nNOS and PLD2;

FIG. 23 shows direct interaction between integrin beta 3, 5 cytosolictail and PLD2;

FIG. 24 shows interaction between integrin beta 3 and PLD2;

FIG. 25 shows an interaction between mTOR and PLD2; and

FIGS. 26A, 26B, and 26C show affinity between PLD1 andphosphoinositide-3-phosphate (PIP3).

DETAILED DESCRIPTION OF THE INVENTION

The terms “polypeptide,” “molecule,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “molecule,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinatedforms, etc. Modifications also include intra-molecular crosslinking andcovalent bonds to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by genes may also be included in a polypeptide.

As used herein, the term “interacting” or “interaction” means that twomolecule domains, fragments or complete molecules exhibit sufficientphysical affinity to each other so as to bring the two “interacting”molecule domains, fragments or molecules physically close to each other.Such interaction can be attained by chemical bond(s) or based solely onphysical affinities. Examples of physical affinities and chemical bondsinclude, but are not limited to, forces caused by electrical chargedifferences, hydrophobicity, hydrogen bonds, van der Waals force, ionicforce, covalent linkages, and combination(s) thereof. The state ofproximity between the interaction domains, fragments, molecules orentities may be transient or permanent, reversible or irreversible. Inany event, it is in contrast to and distinguishable from contact causedby natural random movement of two entities. Typically, although notnecessarily, an “interaction” is exhibited by the binding between theinteraction domains, fragments, molecules, or entities. Examples ofinteractions include specific interactions between antigen and antibody,ligand and receptor, enzyme and substrate, and the like.

As used herein, the term “peptide complex” means a composite unit thatis a combination of two or more peptides formed by interaction betweenthe peptides. Typically, but not necessarily, a “peptide complex” isformed by the binding of two or more peptides together through specificnon-covalent interactions. However, covalent bonds may also be presentbetween the interacting partners. For instance, the two interactingpartners can be covalently crosslinked so that the peptide complexbecomes more stable.

The term “peptide fragment” as used herein means a polypeptide thatrepresents a portion of a peptide. When a peptide fragment exhibitsinteractions with another peptide or peptide fragment, the two entitiesare said to interact through interaction domains that are containedwithin the entities.

The term “isolated peptide complex” means a peptide complex present in acomposition or environment that is different from that found innature—in its native or original cellular or body environment.Preferably, an “isolated peptide complex” is separated from at least50%, more preferably at least 75%, most preferably at least 90%, ofother naturally co-existing cellular or tissue components. Thus, an“isolated peptide complex” may also be a naturally existing peptidecomplex in an artificial preparation or a non-native host cell. An“isolated peptide complex” may also be a “purified peptide complex”,that is, a substantially purified form in a substantially homogenouspreparation substantially free of other cellular components, otherpolypeptides, viral materials, or culture medium, or, when the peptidecomponents in the peptide complex are chemically synthesized, free ofchemical precursors or by-products associated with the chemicalsynthesis. A “purified peptide complex” typically means a preparationcontaining preferably at least 75%, more preferably at least 85%, andmost preferably at least 95%, of a particular peptide complex. A“purified peptide complex” may be obtained from natural or recombinanthost cells or other body samples by standard purification techniques, orby chemical synthesis.

The term “fusion peptide” used herein to mean a non-naturally occurringpeptide having a specified polypeptide molecule covalently linked to oneor more polypeptide molecules that do not naturally link to thespecified polypeptide. Thus, a “fusion peptide” may include twonaturally occurring molecules or fragments thereof linked together by acovalent linkage. A “fusion peptide” may also be-a peptide formed—bycovalently linking two artificial polypeptides together. Typically butnot necessarily, the two or more polypeptides are linked or “fused”together by a peptide bond forming a single non-branched polypeptidechain.

As used herein, the term “modulator” encompasses any compounds that cancause any forms of alteration of the biological activities or functionsof the peptides or peptide complexes, including, e.g., enhancing orreducing their biological activities, increasing or decreasing theirstability, altering their affinity or specificity to certain otherbiological peptides, etc. In addition, the term “modulator” as usedherein also includes any compounds that simply bind PLD, PLD-interactingpeptides, and/or the peptides complexes of the present invention. Forexample, a modulator can be an “interaction antagonist” capable ofinterfering with or disrupting or dissociating peptide-peptideinteraction between PLD or a homologue, fragment or derivative thereofand one or more peptides selected from the group consisting of actin,aldoalse, collapsin response mediator peptide-2 (CRMP-2), phospholipaseC-γ1 (PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1,glucose transporter 4 (GLUT4), mammalian target of rapamycine (mTOR),heat shock protein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxidesynthase (nNOS), integrin beta 3, guanine nucleotide exchange factor—H1(GEF-H1), V-ATPase, phosphoinositide-3-phosphate (PIP3), dopaminetransporter (DAT) or a homologue, fragment or derivative thereof. Amodulator can also be an “interaction agonist” that initiates orstrengthens the interaction between the peptide members of a peptidecomplex of the present invention, or homologues, fragments orderivatives thereof.

The present invention provides an isolated peptide complex comprising: afirst peptide selected from the group consisting of phospholipase D(PLD), a PLD variant, a PLD fragment, and a fusion peptide containingPLD, a PLD variant, or a PLD fragment; and a second peptide selectedfrom the group consisting of actin, aldoalse, collapsin responsemediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT) a variant thereof, a fragment thereof, and a fusion peptidethereof.

The second peptide has a specific binding affinity to PLD, thus forminga peptide complex with PLD. β-Actin, a 43-kDa protein in the rat brain,acts as a major PLD2 direct-binding protein as revealed by peptide massfingerprinting in combination with matrix-assisted laser desorptionionization/time-of-flight mass spectrometry. The region between aminoacids 613 and 723 of PLD2 is required for the direct binding of β-actin,using bacterially expressed glutathione S-transferase fusion proteins ofPLD2 fragments. Purified β-actin is known to potently inhibit bothphosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2activities in a concentration-dependent manner (IC₅₀=5 nM). It had beenpreviously reported that α-actinin inhibited PLD2 activity in aninteraction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversiblemanner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo,J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J.Biol. Chem. 275, 21295-21301). In vitro binding analyses showed thatβ-actin could displace α-actinin binding to PLD2, demonstratingindependent interaction between cytoskeletal proteins and PLD2.Furthermore, ARF1 could steer PLD2 activity in a positive directionregardless of the inhibitory effect of β-actin on PLD2. We also observedthat β-actin regulates PLD1 and PLD2 with similar binding and inhibitorypotencies. Immunocytochemical and co-immunoprecipitation studiesdemonstrated in vivo interaction between the two PLD isozymes and actinin cells. Taken together, such results indicate that the regulation ofPLD by cytoskeletal proteins, β-actin and α-actinin, and ARF1 may playan important role in cytoskeleton-related PLD functions.

Aldolase A also interacts with PLD. To identify the peptides thatinteract with PLD, a peptide overlay assay was performed with fractionsobtained from the sequential column chromatographic separation of ratbrain cytosol using purified PLD2 as a probe. A peptide of molecularweight 40 kDa, which was detected by anti-PLD antibody with overlayingthe purified PLD2, is shown to be aldolase C by peptide-massfingerprinting with matrix-assisted laser desorption/ionization-time-offlight-mass spectrometry (MALDI-TOF-MS). Aldolase A also showed similarbinding property as aldolase C and was co-immunoprecipitated with PLD2in COS-7 cells overexpressing PLD2 and aldolase A. The PH domaincorresponding to amino acids 201-310 of PLD2 was necessary for theinteraction observed in vitro, and aldolase A was found to interact withthe PH domain of PLD2 specifically, but not with other PH domains. PLD2activity was inhibited by the presence of purified aldolase A in adose-dependent manner, and the inhibition by 50% was observed by theaddition of less than micromolar aldolase A. Moreover, the inclusion ofthe aldolase metabolites, such as fructose 1,6-bisphosphate (F-1,6-P) orglyceraldehyde 3-phosphate (G-3-P), resulted in an enhanced interactionbetween PLD2 and aldolase A with a concomitant increase in the potentialability of aldolase A to inhibit PLD2, which suggested the existence ofa possible regulation of the interaction by the change of intracellularconcentrations of glycolytic metabolites.

PLD2 binding activity with a neuronal protein cytosol, from rat brain,was studied. During the fractionation of rat brain cytosol byfour-column chromatography, a 62-kDa PLD2-interacting protein wasdetected by PLD2 overlay assay and identified as collapsin responsemediator protein-2 (CRMP-2), which controls neuronal axon guidance andoutgrowth. Using bacterially expressed glutathione S-transferase fusionproteins, we found that two regions (amino acids 65-192 (the phagocyticoxidase domain) and 724-825) of PLD2 and a single region (amino acids243-300) of CRMP-2 are required for a direct binding of both proteins. Aco-immunoprecipitation study in COS-7 cells also showed an in vivointeraction between CRMP-2 and PLD2. Interestingly, CRMP-2 was found topotently inhibit PLD2 activity in a concentration-dependent manner(IC₅₀=30 nM). Over-expression studies also showed that CRMP-2 is an invivo inhibitor of PLD2 in PC12 cells. Moreover, increasing theconcentration of semaphorin 3A, one of the repulsive axon guidance cues,showed that PLD2 activity could be inhibited in PC12 cells.Immunocytochemistry further revealed that PLD2 is co-localized withCRMP-2 in the distal tips of neurites, its possible action site, indifferentiated PC12 cells. Taken together, CRMP-2 may interact directlywith and inhibit neuronal PLD2, suggesting that this inhibitory mode ofregulation may play a role in neuronal pathfinding during thedevelopmental stage.

PLD interacts with PLC-γ1. The epidermal growth factor (EGF) receptorplays an important role in cellular proliferation, and the enzymaticactivity of phospholipase C γ1 (PLC-γ1) is regarded to be critical forEGF-induced mitogenesis. PLC-γ1 is co-immunoprecipitated with PLD2 inCOS-7 cells. These results of in vitro binding analysis andco-immunoprecipitation analysis in COS-7 cells show that the Srchomology (SH) 3 domain of PLC-γ1 binds to the proline-rich motif withinthe phox homology (PX) domain of PLD2. The interaction between PLC-γ1and PLD2 is EGF stimulation-dependent and potentiates EGF-inducedinositol 1,4,5-trisphosphate (IP3) formation and Ca2+ increase. MutatingPro-145 and Pro-148 within the PX domain of PLD2 to leucines disruptsthe interaction between PLC-γ1 and PLD2 and fails to potentiateEGF-induced IP3 formation and Ca2+ increase. However, neither PLD2 wildtype nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation ofPLC-γ1. These findings suggest that, upon EGF stimulation, PLC-γ1directly interacts with PLD2 and this interaction is important forPLC-γ1 activity.

Although the hydrogen peroxide (H2O2)-dependent activation ofphospholipase D2 (PLD2) in PC12 cells was previously disclosed, theprecise mechanism of PLD1 activation by H2O2 was not revealed (Lee, S.D. et al. (2000) J. Neurochem. 75,1053-1059). In order to findH2O2-dependent PLD2-regulating peptides, the present inventorsimmunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde3-phosphate dehydrogenase (GAPDH) co-immunoprecipitated with PLD2 uponH2O2 treatment. This interaction was found to be direct by in vitroreconstitution of purified GAPDH and PLD2. In vitro studies alsoindicated that PLD2-associated GAPDH was modified on its reactivecysteine residues. Koningic acid, an alkylator of GAPDH on catalyticcysteine residue, also increased interaction between the two peptides invitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2-dependentmodification of GAPDH with 3-aminobenzamide resulted in the inhibitionof the GAPDH/PLD2 interaction and attenuated H2O2-induced PLD2activation in PC12 cells. From the results, we suggest that H2O2modifies GAPDH on its catalytic cysteine residue not only to inactivatethe dehydrogenase activity of GAPDH but also to endow GAPDH with abilityto bind PLD2 and resulting association is involved in the regulation ofPLD2 activity by H2O2. GAPDH is fundamentally a housekeeping enzyme,involved in cellular energy generation. However, growing evidences haveshown that it is not merely a glycolytic enzyme (Sirover, (1999)Biochim. Biophys. Acta 1432, 159-184). It has been shown that GAPDH isinvolved in the early stage of apoptosis (Berry et al., (2000) J.Neurosci. Res. 60, 150-154). A recent study by Colussi et al. showedthat GAPDH inactivation, and hence modification is involved in delayingcellular apoptosis induced by H2O2 (Colussi et al., (2000) FASEB J. 14,2266-2276). In recent years, it was found that the activation of PLD2 byH2O2 might have anti-apoptotic effect (Lee, S. D. et al. (2000) J.Neurochem. 75, 1053-1059). Based on such studies, it could be speculatedthat cellular apoptosis mediated by H2O2 could be modulated by GAPDHmodification and subsequent interaction and activation of PLD2. And suchinteraction and PLD activation are related to anti-apoptotic effect inROS induced apoptosis.

PLD1 interacts with the Akt1. A search of PLD1 binding peptide based onfunctional relationship and similar cellular location identified Akt1 asa binding peptide. Akt, also known-as PKB (protein kinase B), is aserine/threonine molecule kinase that regulates a variety of cellularmolecules implicated in cell proliferation, survival and insulinresponses largely by means of phosphorylation. N-terminal pleckstrinhomology (PH) domain of Akt is important for activation by virtue ofinteraction with PI (3,4,5) P3, triggering the targeting to plasmamembrane where phosphoinositides are generated (Science. May 31,2002;296(5573):1655-7. Akt phosphorylates many pro-apoptotic molecules,inhibiting their activity and enhancing cell survival. Akt is alsoinvolved in cell cycle progression in G1/S and G2/M transition byphosphorylating cell cycle regulators (J Cell Sci. August 2001;114(Pt16):2903-10). Amplification of genes encoding Akt isoforms has beenfound in several types of human cancers (Cell Signal. May2002;14(5):381-95). The interaction of PLD1 with Akt1 provides yetidentified link between PLD1 and the regulation of cell proliferationand survival.

PLD interacts with glucose transporter 4 (GLUT4). Preliminary evidencesshowed that modulation of PLD peptide was involved in the insulindependent glucose transporter 4 (GLUT4) translocation in a variety kindof cells. In this study, we found that PLD1 was co-immunoprecipitatedwith GLUT4 in COS-7 cells overexpressing PLD1 and GLUT4. Thus, weperformed pull down assay using glutathione S-transferase fusionpeptides corresponding to the cytoplasmic domains of the glucosetransporter isoforms with the recombinant PLD1 purified from sf9 cells.PLD1 directly interacted with the central loop of GLUT4 but not withthat of GLUT2. The multiple site of PLD1 including N-, C-terminal regionwas responsible for binding to the central loop of GLUT4. Moreover, inthe response to insulin, GLUT4 was co-localized with PLD1 in the plasmamembranes of the hlRcB cells overexpressing insulin receptor, howeverthe chimeric GLUT4 which was replaced by the loop region of GLUT2 wasnot co-localized with PLD1 in the plasma membrane in regardless toinsulin. These results suggested that the central loop of GLUT 4 wasessential for translocation to the plasma membrane and co-localizationwith PLD1 in insulin dependent manner.

PLD2 interacts with the mTOR. A search of PLD2 binding peptide based onfunctional relationship and similar cellular location identified themTOR (mammalian-target of rapamycin) as a binding peptide (Science. May14, 1999;284(5417):1161-4, EMBO J. Mar. 1, 2000;19(5):1087-97). mTOR isPIKK-related protein kinase which phosphorylates 4E-BP1 and S6K(Science. Jul. 4, 1997;277(5322):99-101, Proc Natl Acad Sci U S A. Feb.17, 1998;95(4):1432-7). Activation of 4E-BP1 and S6K is important fortranslation initiation (Prog Mol Subcell Biol. 2001;26:155-84). Thus,mTOR activity is important for the regulation of cell mass by regulatingtranslation initiation (Genes Dev. Apr. 1, 2001;15(7):807-26). There hasnot been extensive studies on cellular localization of mTOR, however, ithas been reported that it exits in a low-density andcholesterol-enriched membrane fraction. And it is this low-density andcholesterol-enriched membrane fraction in which PLD2 is localizedexclusively. Two binding peptides, raptor and GβL, were reported toregulate mTOR activity with different mechanism compared to that of PLD2(Cell. Jul. 26, 2002;110(2):163-75, Mol Cell. April 2003;11(4):895-904). The role of FAT domain in mTOR was not revealed butsuggested as inhibitory domain for mTOR activity. Binding mappinganalysis and translation assay with PLD2 suggest that PLD2 is yetidentified regulator of mTOR through complex formation. Theidentification of PLD2 as negative regulator of mTOR may promote itsinvolvement in translation initiation.

The present inventors investigated PLD2-binding proteins byco-immunoprecipitation assay from PLD2-overexpressed PC12 cells. Proteinhaving a molecular weight of 70 kDa was specifically co-precipitatedwith PLD2 and identified as Heat Shock Protein (HSP70) usingpeptide-mass fingerprinting with matrix-assisted laserdesorption/ionization (MALDI) mass spectrometer. The amino acid residues120-428 of HSP70 containing ATPase domain and amino acid residues186-308 of PLD2 containing PH domain were responsible for theinteraction between PLD2 and HSP70. Purified HSP70 potently inhibitedPLD2 activity (IC₅₀=200 nM) and the inhibition was dependent on itsinteraction with PLD2. Furthermore, attenuated activity of PLD2 by HSP70was stimulated up to 5-fold by ARF without any effect on the interactionbetween PLD2 and HSP70. These results suggest that PLD2 possesses thepotential capacity of ARF responsiveness by the attenuation of highbasal activity by HSP70. In response to many metabolic disturbances andinjuries including stroke, neurodegenerative disease, epilepsy andtrauma, the cell mounts a stress response with induction of a variety ofproteins, most notably the 70 kD heat shock protein (Adv Exp Med Biol.2002; 513: 281-99). Several reports have now shown that selectiveoverexpression of HSPsp70 leads to protection in several differentmodels of nervous system injury. Accordingly, the interaction betweenPLD and HSPsp 70 and an inhibitor thereof are studied.

The present inventors identified dynamin as a PLD2-interacting peptidein rat brain. Dynamin is a GTPase family member and has been implicatedin the formation of nascent vesicles in both the endocytic and secretorypathways. Dynamin was found to interact with PLD2 in a GTP-dependentmanner, whereas DYN-K44A, a dominant negative mutant of dynamin, did notinteract with PLD2. The PLD2-interacting site on dynamin was identifiedas the GTPase domain and the dynamin-interacting site on PLD2 overlappedwith the PX domain of PLD2. Interaction between dynamin and PLD2 wastransiently increased by treatment with EGF in a time dependent manner,and this was found to correlate with the activation of PLD2.Furthermore, the overexpression of DYN-K44A repressed EGF-induced PLDactivity and MAP kinase phosphorylation in COS-7 cells. These resultssuggest that the GTP-dependent interaction of PLD2 with dynamin wouldlikely play an important role in EGF-induced PLD activation and cellsignaling. One of the most evident correlation between the severity ofdementia due to Alzheimer's disease and its rational cause is the lossof neocortical and hippocampal synapses along with other changes. Axonalterminals are dependent on axoplasmic flow, and that function requiresmicrotubules and the motor proteins kinesin, dynein and dynamin to beintact. (J Neural Transm Suppl. 1998;53:141-5). The present inventorspredicted that PLD could interact with dynamin in a GTP-dependentmanner. In return, the interaction between Dynamin and PLD may regulateconditions related to Alzheimer's disease.

The present inventors investigated PLD-binding proteins obtained fromrat brain extract, and identified a 67-kDa protein as Munc-18-1 bypeptide-mass fingerprinting. The direct association between the twoproteins was confirmed using purified proteins, and the binding site wasdetermined as the phox homology domain of PLD and multiple sites ofMunc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro.Moreover, the co-transfection of Munc-18-1 and PLD into COS-7 cells wasfound to inhibit PLD activity, indicating that Munc-18-1 is anintracellular inhibitor of PLD activity. Munc-18-1 was co-precipitatedwith PLD in a basal state, and PLD2 was colocalized at plasma membraneof COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1from PLD when EGF activated PLD. Translocation of Munc-18-1 frommembrane to cytosol after EGF treatment was also observed byimmunocytochemical analysis. The dissociation of Munc-18-1 from theintrinsic PLD and the activation of PLD by EGF were also observed inchromaffin cells. These results suggest that Munc-18-1 is a potentnegative regulator of basal PLD activity and that EGF stimulationabolishes their interaction and activates PLD. Munc-18 proteins arerelated to vesicle trafficking and exocytosis. They are involved ininsulin secretion (J Biol Chem. Dec. 29, 2000;275(52):41521-7) and GLUT4translocation (J Biol Chem. Feb. 9, 2001;276(6):4063-9.). PLD is alsoinvolved in these processes. Insulin secretion is known as a determinantof diabetes 1 and GLUT4 translocation is related to diabetes 2. From theresults of binding between PLD and Munc-18, it was observed that thisinteraction is dependent on the extracellular signals and regulates PLDactivity. From these results, we can suggest that interaction betweenPLD and Munc-18-1 can be related in diabetes and by elucidating suchactivity, we can have a new insight in the specific molecular mechanismof diabetes.

The cholinergic regulation of phospholipase D activity has been widelystudied in many cell types. However, whereas activation mechanisms ofCCh-induced PLD activation are well known, down regulation mechanisms ofPLD activity have not yet been elucidated. To investigate the downregulation mechanism of CCh-induced PLD activation, binding peptides onPLD activity down regulated status were sought. As a result, we foundthat a 55-kDa molecule in the M3 acetylcoline receptor expressed COS-7cells, α-, β-tubulin dimmer, specifically interact with PLD2 in timedependent manner. We also observed that tubulin directly interacts withamino acid 65-192 and 475-612 region of PLD and inhibits its activity invitro. Tubulin constitutes microtubules, a major component ofcytoskeleton. Tubulin exists principally in two forms, either ascytosolic soluble tubulin heterodimers consisting of various α- andβ-tubulin isotypes or as insoluble assembled tubulin polymers(microtubules). To test the PLD regulation through the changes indynamics of tubulin, a microtubule structure regulating agent was used.In COS-7 cells, interaction of PLD2 with tubulin increased withnocodazole, a microtubule depolymerizing agent, and decreased withtaxol, a microtubule stabilizing agent. In this condition, compared tomuscarinic response, nocodazole pretretment inhibits the CCh-inducedPLD2 activity while taxol-pretreated cells increased PLD2 activity.Furthermore, in endogeneously muscarinic receptor expressed PC12 cells,a specific and time-dependent association of tubulin with PLD2 wasobserved after 1 min when the PLD2 activity was in the off-status. Andimmunocytochemistry further revealed that tubulin translocated to plasmamembrane and colocalized with PLD2 after CCh stimulation. Takentogether, the results indicate that an increase of monomeric tubulinconcentration down regulates carbachol-stimulated PLD activity.Microtubules are involved in many cellular functions such as cell cycle,endocytosis, exocytosis, vesicle trafficking, and maintaining cellshape. Especially, in neuronal cell, microtubule is important forneuronal differentiation, neurite outgrowth, neurite retraction and axonguidance. So, it is possible for this PLD activity regulation by changesin microtubule dynamics to involve these processes. In fact, it isnoteworthy that activity shapes the structure of neurons and theircircuits. Synaptic activation is shown to produce rapid input-specificchanges in dendritic structure (Maletic-Savatic et al., 1999). And thepossibility exists that the neurotransmitter-evoked recruitment oftubulin to the membrane assists with this process. In fact, it has beensuggested previously that the synaptic activity-controlled balancing ofmonomeric, dimeric, and polymeric forms of actin and tubulin mightunderlie the changes in spine shape (Van Rossum and Hanisch, 1999).

PLD interacts with nNOS (neuronal nitric oxide synthase) in rat brain.Co-immunoprecipitation assay with anti-PLD antibody in rat brain wasperformed and PLD-binding peptide with relative molecular weights of 160kDa was found. The band was analyzed by matrix-assisted laserdesorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Asearch for these masses in a comprehensive sequence database showed that17 masses (red sequences) matched the calculated tryptic peptide massesof nNOS with an accuracy of <0.1 Da. Nitric oxide (NO), synthesized bythe enzyme NO syntase (NOS), is a potent cell signaling and vasodilatormolecule that plays important and diverse roles in biological processesincluding the control of vascular tone and renal sodium excretion.Because of its potent and diverse biological effects, NO production byNOS is under complex and tight control. nNOS can be regulated byinteraction with binding partners such as calmodulin, Hsp90, PIN,caveolin-3, PDZ domain containing molecules (PSD-95, PSD-93,1-syntrophin, CAPON, phosphofurctokinase-M). Calmodulin and Hsp90activate, and PIN inhibits NOS activity through direct interaction. nNOSmore strongly interacts with PLD2 than PLD1 in COS-7 cells. When nNOSwas co-immunoprecipited with anti-PLD antibody and PLDs wereco-immunoprecipitated with anti-FLAG antibody, nNOS interacts with PLD2in EGF-dependent manner in COS-7 cells. It was reported that nNOS wasstimulated by binding of Ca²⁺-calmoduin according to increasedintracellular Ca²⁺ level. It was also reported that EGF can stimulatePLD activity in a time and dose-dependent manner and increaseintracellular Ca²⁺ level in COS-7 cells. Therefore, we verifiedinteraction between PLD2 and nNOS after EGF stimulation in COS-7 cells.PLD2 maximally interacts-with nNOS at 0.5-1 min after EGF stimulation.

We showed a specific interaction of PLD with integrin 3, 5 but not 1,both in vitro and in vivo by immunoprecipitation assay. PLD interactswith C-terminal of integrin 5 cytosolic tail. Integrins are alpha-betaheterodimer, and each alpha-beta combination has its own ligand bindingspecificity and signaling properties. Most integrins recognize severalextracellular matrix molecules according to alpha-beta combination. Andthen, integrin is the place in which several signals are integrated, andinvolved in many signaling pathway. Many numbers of signaling enzymesand adaptor molecules are regulated by integrin control cell survival,proliferation, motility and differentiation so. It is possible forspecific interaction of integrin and PLD to involve in these processespecially in cancer and metastasis process.

PLD interacts with GEF-H1. Flag tagged GEF-H1 is coimmunoprecipitatedwith each of PLD isozymes, especially PLD2 stronger than PLD1, in COS 7cell. GEF-H1 is a microtubule-interacting peptide, identified byhomology to guanine nucleotide exchange factors (GEFs) in a screen of aHeLa cell cDNA library (Ren et al., J Biol Chem, 273(52):34954-60(1998)). GEF-H1 contains a N-terminal zinc finger domain, a C-terminalcoiled-coil domain; immunocytochemistry experiments reveal that thesedomain are responsible for colocalization of GEF-H1 with microtubules.These recent reports suggested that the GEF activity of GEF-H1 wasregulated by association with microtubules (Krendel et al., Nat CellBiol, Apr;4(4):294-301 (2002)). GEF-H1 also contains a Dbl-type GEFdomain, in tandem with a pleckstrin homology domain, a motif typicallyresponsible for exchange of GTP and GDP. GEF-H1 stimulates guaninenucleotide exchange of Rho (known regulators of the cytoskeleton),converting inactive GDP-bound Rho into active GTP-bound form whichinteracts many effector molecules. Phospholipase D (PLD) is one of thoseeffectors, and plays a role in hydrolysis of phosphatidylcholine to formphosphatidic acid (PA) and choline (Exton, J. H. (1999) Biochim.Biophys. Acta 1439, 121-133). Another postulated role for PLD is in theregulation of the actin cytoskeleton; PLD activity is required for actincytoskeleton rearrangement to form stress fibers (Exton, J. H., Mol CellBiol. 2001, 21(12):4055-66).

PLD interacts with V-ATPase subunit A. To investigate anotherPLD-interacting peptide, we tried to co-immunoprecipiate it withanti-PLD antibody from rat brain cytosol. By molecule peptide-massfingerprinting, V-ATPase subunit A is identified as a PLD interactingpeptide. The vacuolar (H⁺)-ATPase (V-ATPase) is a multisubunit enzymethat facilitates the acidification of intracellular compartments ineukaryotic cells and plays an important role in receptor-mediatedendocytosis, renal acidification, bone resorption and activation of acidhydrolases (Forgac, M. (2002) Nat. Rev. Mol. Cell Biol. 3, 94-103).Among the subunits of V-ATPase, subunit A is a 68 kD molecule which hasa role in ATP hydrolysis together with another subunit B (Steves, T. H.,and Forgac, M. (1997) Annu. Rev. Cell Dev. Biol. 13, 779-808). Toconfirm the interaction of PLD and V-ATPase subunit A, Flag taggedV-ATPase subunit A is coimmunoprecipitated with each of PLD isozymes,especially PLD2 stronger than PLD1, in COS 7 cell. Since PLD has beenimplicated in vesicle formation and receptor-mediated endocytosis(Yingjie Shen (2001) Mol Cell Biol. 21(2), 595-602.), PLD might play anew role in regulating receptor endocytosis with the interaction of theV-ATPase.

In terms of domain function of PLD, molecular mechanisms involved in theactivity regulation are still unclear. Especially, the involvement ofphox homology (PX) domain in targeting and regulation of PLD has notbeen elucidated. The properties of PLD PX-domains in terms of thephosphoinositide binding and the regulation of PLD activity had beenstudied. Interestingly, the PX domain of PLD1, but not that of PLD2,specifically interacted with phosphatidylinositols-3,4,5-trisphosphate(Ptdlns(3,4,5)P₃) but not with any other phosphoinositides in bothprotein-lipid overlay and liposome-binding assays. Mutation of conservedarginine to lysine (R179K) or alanine (R179A) in PX domain of PLD1disrupted Ptdlns(3,4,5)P₃ binding. EGFP-PLD1 PX domain, but neither theR179K nor R179A mutant, was localized in membrane fraction only when theconstitutively active form of phosphoinositide 3-kinase (p110-CAAX) wasco-transfected in COS-7 cells, suggesting that the PX domain mayinteract with Ptdlns(3,4,5)P₃ in cells. PLD1 activity was stimulated bythe addition of Ptdlns(3,4,5)P₃ in vitro. The PLD1 mutants (PLD1(R179K)and PLD1(R179A)) were not responsive on Ptdlns(3,4,5)P₃. Co-transfectionof p110-CAAX with wild-type PLD1 but not with PLD1(R179K) or PLD1(R179A)significantly increased PLD activity in COS-7 cells. Moreover,insulin-stimulated PLD activity was inhibited by LY 294002, which is aspecific inhibitor of phosphoinositide 3-kinase. PLD-inducedextracellular signal-regulated kinase (ERK) phosphorylation was observedin wild-type expressing cells but not in cells expressing PLD1(R179K)and PLD1(R179A). Our results suggest that the PLD1 PX domain enablesPLD1 to mediate signal transduction through ERK by providing directbinding site for Ptdlns(3,4,5)P₃ and activating PLD1.

PLD plays an important role in the cellular signal transduction and itsactivity is thought to mediate vesicle trafficking in cells by producingphosphatidic acid (PA), which recruits AP-2, an adaptor protein forclathrin. PLD directly interacts with Na⁺/Cl⁻-dependent dopaminetransporter (DAT) which is critical in terminating dopaminergictransmission by removing the transmitter away from the synapse. In humanembryonic kidney 293 cells, PLD induces DAT degradation in an activitydependent manner. Furthermore, binding deficient mutant of PLD showed noexpression-inhibiting effect on DAT, indicating that activity and directinteraction are important in PLD-mediated down regulation of DAT.Inhibitor studies indicated that PLD shifts the trafficking route of DATfrom the steady-state recycling between plasma membrane and endosome tolysosomal degradation pathway. These findings suggest a potential rolefor PLD activity and interaction in the redistribution and degradationof DAT.

Accordingly, the present invention provides peptide complexes formedbetween PLD and one or more PLD-interacting peptides selected from thegroup consisting of actin, aldoalse, collapsin response mediatorpeptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT). Further, one or more of the interacting molecule members of apeptide complex of the present invention include a variant thereof, afragment thereof, and a fusion peptide containing such, in addition to anative peptide.

The present invention also provides peptide complexes of the foregoingwherein it comprises a PLD and another peptide selected from the groupconsisting of actin, aldoalse, collapsin response mediator peptide-2(CRMP-2), phospholipase C-γ1 (PLC-γ1), glyceraldehyde-3-phosphatedehydrolase (GAPDH), Akt1, glucose transporter 4 (GLUT4), mammaliantarget of rapamycine (mTOR), heat shock protein 70 (hsp70), dynamin,munc 18, tubulin, n-nitric oxide synthase (nNOS), integrin beta 3,guanine nucleotide exchange factor—H1 (GEF-H1), V-ATPase,phosphoinositide-3-phosphate (PIP3), dopamine transporter (DAT).

As described above, individual peptide fragments involved in thespecific peptide-peptide interactions have been discovered.-Accordingly, peptide fragments containing the amino acid sequence ofthe identified regions or variants thereof can be used in forming thepeptide complexes of the present invention. In addition, fragmentscapable of interacting with PLD can also be provided by the combinationof molecular engineering of a nucleic acid encoding a PLD-interactingpeptide and a method for testing peptide-peptide interaction.

In an embodiment of the peptide complex of the present invention, two ormore interacting partners (PLD and one or more peptides selected fromthe group consisting of actin, aldoalse, collapsin response mediatorpeptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT), variants thereof, and fragments thereof) are directly fusedtogether, or covalently linked together through a peptide linker,forming a hybrid peptide having a single unbranched polypeptide chain.Thus, the peptide complex may be formed by “intramolecular” interactionsbetween two portions of the hybrid peptide. Again, one or both of thefused or linked interacting partners in this peptide complex may be anative peptide or variants or fragments of a native peptide.

The above-described peptide complexes may further include any additionalcomponents, e.g., other peptides, nucleic acids, lipid molecules,monosaccharides or polysaccharides, ions, etc.

The isolated peptide complex of the present invention may be prepared byvariety of methods. Specifically, the peptide complex may be isolateddirectly from an animal tissue sample. Alternatively, the peptidecomplex may be purified from recombinant host cells that express themembers of the peptide complex. As will be apparent to a skilled personin the art, a peptide complex may be prepared from a tissue sample orrecombinant host cells by co-immunoprecipitation using an antibody thatis immuno-reactive with an interacting peptide partner, or preferably anantibody selectively immuno-reactive with the peptide complex as will bediscussed in detail below.

The antibodies can be monoclonal or polyclonal. Co-immunoprecipitationis a commonly used method in the art for isolating or detecting boundpeptides. In such procedure, generally a serum sample or tissue or celllysate is admixed with a suitable antibody. The peptide complex bound tothe antibody is precipitated and washed. The bound peptide complexes arethen eluted.

Alternatively, immunoaffinity chromatography and immunoblotingtechniques may also be used to isolate peptide complexes from nativetissue samples or recombinant host cells using an immunoreactiveantibody with an interacting peptide partner, or preferably an antibodyselectively immunoreactive with the peptide complex. Immunoblotting,crude peptide samples from a tissue sample extract or recombinant hostcell lysate are fractionated by polyacrylamide gel electrophoresis(PAGE) and then transferred to a membrane, e.g., nitrocellulose.Components of the peptide complex can then be located on the membraneand identified by a variety of techniques, e.g., probing with specificantibodies.

In an embodiment, the peptide complex of the present invention may beprepared from tissue samples or recombinant host cells or other suitablesources by peptide affinity chromatography or affinity blotting. Thatis, one of the interacting peptide partners is used to isolate the otherinteracting peptide partner(s) by binding affinity and forming peptidecomplexes. Thus, an interacting peptide partner prepared by purificationfrom tissue samples or by recombinant expression may be bound covalentlyor non-covalently to a matrix. The tissue sample extract or cell lysatefrom the recombinant cells can then be contacted with the bound peptideon the matrix. In affinity blotting, crude peptide samples from thetissue sample or recombinant host cell lysate can be fractionated bypolyacrylamide gel electrophoresis (PAGE) and then transferred to amembrane, e.g., nitrocellulose. The purified interacting peptide memberis then bound to its interacting peptide partner(s) on the membraneforming peptide complexes, which are then isolated from the membrane.

It will be apparent to one skilled in the art that any recombinantexpression methods may be used in the present invention for purposes ofexpressing the peptide complexes or individual interacting peptides.Generally, a nucleic acid encoding an interacting peptide member can beintroduced into a suitable host cell. For purposes of forming arecombinant peptide complex within a host cell, nucleic acids encodingtwo or more interacting peptide members are introduced into the hostcell.

Typically, the nucleic acids, preferably in the form of DNA, areincorporated into a vector to form expression vectors capable ofdirecting the production of the interacting peptide member(s) onceintroduced into a host cell. Many types of vectors can be used for thepresent invention.

An epitope tag coding sequence for detection and/or purification of theexpressed peptide can also be operably linked to the DNA encoding aninteracting peptide member such that a fusion peptide is expressed.Examples of useful epitope tags include, but are not limited to,influenza virus hemagglutinin (HA), Flag, polyhistidine (6× His), GST,and the like. Peptides with polyhistidine tags can be easily detectedand/or purified with Ni affinity columns, while specific antibodiesimmunoreactive with many epitope tags are generally commerciallyavailable. The expression vectors may also contain components thatdirect the expressed peptide in an extracellular or a particularintracellular compartment. Signal peptides, myristoylation signals,palmitoylation signals, and transmembrane sequences are example ofoptional vector components that can determine the destination ofexpressed peptides. When it is desirable to express two or moreinteracting peptide members in a single host cell, the DNA fragmentsencoding the interacting peptide members may be incorporated into asingle vector or different vectors.

The expression vectors can then be introduced into the host cells by anytechniques known in the art, e.g., by direct DNA transformation,electroporation, viral infection, lipofection, and the like. Theexpression of the interacting peptide members may be transient orstable. The expression vectors can be maintained in host cells in anextrachromosomal state, i.e., as self-replicating plasmids or viruses.Alternatively, the expression vectors can be integrated into chromosomesof the host cells by conventional techniques such as selection of stablecell lines or site-specific recombination. In stable cell lines, atleast the expression cassette portion of the expression vector isintegrated into a chromosome of the host cells.

The vector construct can be designed to be suitable for expression invarious host cells, including but not limited to bacteria, yeast cells,plant cells, insect cells, and mammalian cells. Methods for preparingexpression vectors for expression in different host cells should beapparent to a skilled artisan.

Variants and fragments of the native interacting peptide members canalso be easily expressed using the recombinant methods described above.For example, to express a peptide fragment, the DNA fragmentincorporated into the expression vector can be selected such that itonly encodes the peptide fragment. Likewise, a specific hybrid peptidecan be expressed using a recombinant DNA encoding the hybrid peptide.Similarly, a variant peptide may be expressed from a DNA sequenceencoding the variant peptide. A variant-encoding DNA sequence may beobtained by manipulating the native peptide-encoding sequence usingrecombinant DNA techniques. For this purpose, random or site-directedmutagenesis can be conducted using techniques generally known in theart.

To make variants, for example, the amino acid sequence of a nativeinteracting peptide member may be changed in predetermined manners bysite-directed DNA mutagenesis to create or remove consensus sequencesfor, e.g., phosphorylation by protein kinases, glycosylation,ribosylation, myristolation, palmytoylation, ubiquitination, and thelike. Alternatively, non-natural amino acids can be incorporated into aninteracting peptide member during the synthesis of the peptide inrecombinant host cells. In addition, variants of the native interactingpeptide members of the present invention can also be prepared bychemically linking certain moieties to amino acid side chains of thenative peptides. If desired, variants thus generated can be tested todetermine whether they are capable of interacting with their intendedpartners to form peptide complexes. Testing can be conducted by othermethods known in the art for detecting peptide-peptide interaction.

A hybrid peptide as described above having PLD or a variant or fragmentthereof covalently linked by a peptide bond or a peptide linker to apeptide selected from the group consisting of actin, aldoalse, collapsinresponse mediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp7o), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT), a variant thereof, and a fragment thereof, can be expressedrecombinantly from a chimeric nucleic acid, e.g., a DNA or mRNA fragmentencoding the fusion peptide.

The modulators selected in accordance with the screening method of thepresent invention can be effective in modulating the functions oractivities of PLD, a PLD-interacting peptide, or the peptide complexesof the present invention. Any test compounds may be screened by thescreening method of the present invention to select modulators of PLD, aPLD-containing peptide complex and/or a PLD-interacting peptide of thepresent invention.

The test compounds may be screened to identify compounds capable ofbinding the peptide complexes or interacting peptide members thereof inaccordance with the present invention. For this purpose, a test compoundis contacted with a peptide complex or an interacting peptide memberthereof under conditions and for a time sufficient to allow specificinteraction between the test compound and the target components to occurand thus binding of the compound to the target forms a complex.Subsequently, the binding event is detected.

Various screening techniques known in the art may be used in the presentinvention. The peptide complexes and the interacting peptide membersthereof may be prepared by any suitable methods, e.g., by recombinantexpression and purification. The peptide complexes and/or interactingpeptide members thereof (both are referred to as “target” hereinafter inthis section) may be free in solution. A test compound may be mixed witha target, to form a liquid mixture. The compound may be labeled with adetectable marker. Upon mixing under suitable conditions, the bindingcomplex of the compound and the target may be co-immunoprecipitated andwashed. The compound in the precipitated complex may be detected basedon the marker on the compound.

In an embodiment, a peptide complex used in the screening methodincludes a fusion peptide, which is formed by fusion of two interactingpeptide members or fragments or interaction domains thereof. The fusionpeptide may also be designed such that it contains a detectable epitopetag fused thereto. Suitable examples of such epitope tags includesequences derived from, e.g., influenza virus hemagglutinin (HA),polyhistidine (6.times.His), c-myc, MBP, GST, and the like.

Test compounds may be also screened to identify compounds capable ofdissociating the peptide complexes identified in accordance with thepresent invention. Thus, for example, a PLD-containing peptide complexcan be contacted with a test compound and the peptide complex can bedetected. Conversely, test compounds may also be screened to identifycompounds capable of enhancing the interaction between PLD and aPLD-interacting peptide or stabilizing the peptide complex formed by thetwo or more peptides. The method of screening would be carried out in asimilar manner as described above. For example, the presence of aparticular peptide complex can be detected by an antibody selectivelyimmunoreactive with the peptide complex. Thus, after incubation of thepeptide complex with a test compound, an immunoprecipitation assay canbe conducted with the antibody. If the test compound disrupts thepeptide complex, then the amount of immunoprecipitated peptide complexin this assay will be significantly less than that in a control assay inwhich the same peptide complex is not contacted with the test compound.Similarly, the interaction between two peptides, which is to beenhanced, may be incubated together with the test compound. Thereafter,the peptide complex may be detected by the selectively immunoreactiveantibody. The amount of peptide complex may be compared to that formedin the absence of the test compound. Various other detection methods maybe suitable in the dissociation method, as will be apparent to oneskilled in the art.

The screening methods of modulators may be carried out both in vivo andin vitro environments. For example, any in vivo or in vitro assays knownin the art to be useful in identifying modulators capable ofstrengthening or interfering with the stability of the peptide complexesof the-present invention may be used.

The screening assays of the present invention are useful in identifyingcompounds capable of interfering with or disrupting or dissociatingpeptide-peptide interactions between PLD, or a variant thereof, and apeptide selected from the group consisting of actin, aldoalse, collapsinresponse mediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT) and a variant thereof. For example, PLD, or a variant thereof, andits interacting partners, or variants thereof, are believed to play arole in budding, intracellular vesicle trafficking and vacuolar peptidesorting, formation of multivesicular bodies, exocytosis, endocytosis,tumorigenesis and cell transformation, and autoimmune response, neuronaldisorder and thus are involved in cancer and autoimmune diseases. It maybe possible to ameliorate or alleviate the diseases or disorders in apatient by interfering with or dissociating normal interactions betweenPLD and one of actin, aldoalse, collapsin response mediator peptide-2(CRMP-2), phospholipase C-γ1 (PLC-γ1), glyceraldehyde-3-phosphatedehydrolase (GAPDH), Akt1, glucose transporter 4 (GLUT4), mammaliantarget of rapamycine (mTOR), heat shock protein 70 (hsp70), dynamin,munc 18, tubulin, n-nitric oxide synthase (nNOS), integrin beta 3,guanine nucleotide exchange factor—H1 (GEF-H1), V-ATPase,phosphoinositide-3-phosphate (PIP3), dopamine transporter (DAT).Alternatively, if the disease or disorder is associated with increasedexpression of PLD and/or one of the PLD-interacting peptides inaccordance with the present invention, then the disease may be treatedor prevented by weakening or dissociating the interaction between PLDand the PLD-interacting peptide in patients. In addition, if a diseaseor disorder is associated with variants of PLD and/or one of thePLD-interacting peptides that lead to strengthened peptide-peptideinteraction therebetween, then the disease or disorder may be treatedwith a compound that weakens or interferes with the interaction betweenthe variant of PLD and/or the PLD-interacting peptide(s).

In a screening method for modulators of an interaction, PLD (or fragmentthereof, or a variant of PLD (or fragment thereof, and a PLD-interactingpeptide (or a variant or fragment thereof, or a variant of aPLD-interacting peptide (or fragment thereof), are used as test peptidesexpressed in the form of fusion peptides as described above for purposesof checking interaction of PLD and PLD-interacting peptide.

In an embodiment, a counterselectable marker is used as a reporter suchthat a detectable signal (e.g., appearance of color or fluorescence) ispresent only when the test compound is capable of interfering with theinteraction between the two test peptides.

In another embodiment, the interaction or interaction change between thefirst peptide and the second peptide is determined in a host cell.

The screening assays of the present invention can also be used inidentifying compounds that trigger or initiate, enhance or stabilizepeptide-peptide interactions between PLD, or a variant thereof, and apeptide selected from the group consisting of actin, aidoalse, collapsinresponse mediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT), and a variant thereof. For example, if a disease or disorder isassociated with decreased expression of PLD and/or a member of selectedfrom the group consisting of actin, aldoalse, collapsin responsemediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT), then the disease or disorder may be treated or prevented bystrengthening or stabilizing the interaction between PLD and thePLD-interacting member in patients. Alternatively, if a disease ordisorder is associated with variant of PLD and/or variants of aPLD-interacting peptide that lead to weakened or abolishedpeptide-peptide interaction therebetween, then the disease or disordermay be treated with a compound that initiates or stabilizes theinteraction between the variants of PLD and/or the variants ofPLD-interacting peptide(s).

Thus, a screening assay can be performed in the same manner as describedabove, except that a positively selectable marker is used. For example,PLD (or a variant or fragment thereof), or a variant of PLD (or avariant or fragment thereof), and a peptide selected from the groupconsisting-of actin, aldoalse, collapsin response mediator peptide-2(CRMP-2), phospholipase C-γ1 (PLC-γ1), glyceraldehyde-3-phosphatedehydrolase (GAPDH), Akt1, glucose transporter 4 (GLUT4), mammaliantarget of rapamycine (mTOR), heat shock protein 70 (hsp70), dynamin,munc 18, tubulin, n-nitric oxide synthase (nNOS), integrin beta 3,guanine nucleotide exchange factor—H1 (GEF-H1), V-ATPase,phosphoinositide-3-phosphate (PIP3), dopamine transporter (DAT) (or avariant or fragment thereof), or a variant of a peptide selected fromthe group consisting of actin, aldoalse, collapsin response mediatorpeptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT) (or a variant or fragment thereof), are used as test peptidesexpressed in the form of fusion peptides as described above for purposesof pull down assay. The fusion peptides are expressed in host cells andare allowed to interact with each other in the presence of one or moretest compounds.

Generally, a control assay is performed in which the above screeningassay is conducted in the absence of the test compound. The result isthen compared with that obtained in the presence of the test compound.

In an embodiment, the screening method further comprises generating adata set defining one or more selected test compounds, wherein the dataset is embodied in a transmittable form.

As described above, the interactions between PLD and the PLD-interactingpeptides suggest that these peptides and/or the peptide complexes may beinvolved in common biological processes and disease pathways. Thus, onemay modulate such biological processes or treat diseases by modulatingthe functions and activities of PLD, a PLD-interacting peptide, and/or apeptide complex comprising a combination of these peptides. As usedherein, modulating PLD, a PLD-interacting peptide, or a peptide complexcomprising a combination of these peptides means altering (enhancing orreducing) the activities of the peptides or peptide complexes, e.g.,increasing the concentrations of PLD, a PLD-interacting peptide or apeptide complex comprising a combination of these peptides, enhancing orreducing their biological activities, increasing or decreasing theirstability, altering their affinity or specificity to certain otherbiological peptides; etc. For example, a PLD-containing peptide complexof the present invention or its members thereof may be involved inbudding, intracellular vesicle trafficking and vacuolar peptide sorting,formation of multivesicular bodies, endocytosis, tumorigenesis and celltransformation, and proliferation and may be associated with diseasesand disorders such as neurodegenerative diseases, cancer and diabetes,and the present invention may be used in determining the effect of anaberration in a particular PLD-containing complex or an interactingmember thereof on the above specified occurrences. In addition, it isalso possible to determine, using the same assay methods, the presenceor absence of an association between a PLD-containing complex or aninteracting member thereof and a physiological disorder or disease suchas cancer and autoimmune diseases or predisposition to a physiologicaldisorder or disease.

In accordance with this aspect of the present invention, methods areprovided for modulating (promoting or inhibiting) a PLD-containingprotein complex or interacting member thereof. The human cells can be inin vitro cell or tissue cultures.

In one embodiment, the concentration of a PLD-containing protein complexof the present invention is reduced in the cells. Various methods can beemployed to reduce the concentration of the protein complex. The proteincomplex concentration can be reduced by interfering with the interactionbetween the interacting members. For example, compounds capable ofinterfering with interactions between PLD and a protein selected fromthe group consisting of actin, aldoalse, collapsin response mediatorpeptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT) can be administered to the cells in vitro or in vivo. Suchcompounds can be compounds capable of binding PLD or the proteinselected from the group consisting of actin, aldoalse, collapsinresponse mediator peptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT). They can also be antibodies immunoreactive with the PLD or theprotein selected from the group consisting of actin, aldoalse, collapsinresponse mediator peptide-2-(CRMP-2), phospholipase C-γ1 (PLC-γ₁),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT). Also, the compounds can be small peptides derived from aPLD-interacting protein or mimetics thereof capable of binding PLD, orsmall peptides derived from TPLD protein or mimetics thereof capable ofbinding a protein selected from the group consisting of actin, aldoalse,collapsin response mediator peptide-2 (CRMP-2), phospholipase C-γ1(PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT).

In various embodiments described above, preferably, the concentrationsor activities of both PLD protein and a PLD-interacting protein arereduced or inhibited.

In yet another embodiment, an antibody selectively immunoreactive with aprotein complex having PLD interacting with a protein selected from thegroup consisting of actin, aldoalse, collapsin response mediatorpeptide-2 (CRMP-2), phospholipase C-γ1 (PLC-γ1),glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT) is administered to cells in vitro or in human bodies to inhibitthe protein complex activities and/or reduce the concentration of theprotein complex in the cells or patient.

In one aspect of the present invention, methods are provided forreducing in cells or tissue the concentration and/or activity of aprotein complex identified in accordance with the present invention thatcomprises PLD and one or more members of the group consisting of actin,aldoalse, collapsin response mediator peptide-2 (CRMP-2), phospholipaseC-γ1 (PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1,glucose transporter 4 (GLUT4), mammalian target of rapamycine (mTOR),heat shock protein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxidesynthase (nNOS), integrin beta 3, guanine nucleotide exchangefactor—H1-(GEF-H1), V-ATPase, phosphoinositide-3-phosphate (PIP3),dopamine transporter (DAT). In addition, methods are also provided forreducing in cells or tissue the concentration and/or activity of aPLD-interacting protein selected from the group consisting of actin,aldoalse, collapsin response mediator peptide-2 (CRMP-2), phospholipaseC-γ1 (PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1,glucose transporter 4 (GLUT4), mammalian target of rapamycine (mTOR),heat shock protein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxidesynthase (nNOS), integrin beta 3, guanine nucleotide exchange factor—H1(GEF-H1), V-ATPase, phosphoinositide-3-phosphate (PIP3), dopaminetransporter (DAT). By reducing the concentration of protein complexand/or the PLD-interacting protein concentration(s) and/or inhibitingthe functional activities of the protein complex and/or thePLD-interacting protein(s), the diseases involving such protein complexor PLD-interacting protein(s) may be treated or prevented.

The present invention is further illustrated and described by thefollowing examples, which should not be taken to limit the scope of theinvention.

EXAMPLE 1 Interaction between PLD2 and β-actin

(1) Co-Precipitation of PLD-2-Binding Proteins from Rat Brain Extracts

Hexa-histidine (His₆)-tagged PLD2 was purified from detergent extractsof baculovirus-infected sf9 cells by chelating-Sepharose affinity columnchromatography according to J. H. Kim et. al., FEBS Lett. 454,42-46,1999. Rat brains (3 g) were homogenized in homogenation buffer (20mM Tris/HCl, pH 7.5,1 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and 150 mM NaCl)using a polytron homogenizer. After centrifugation at 100,000×g for 1hour at 4° C., the resulting supernatant was used to investigatepotential PLD2-binding partners. Protein concentrations in the brainextract were determined using the method according to M. M. Bradford,Anal. Biochem. 72, 248-254, 1976.

Affinity-purified anti-PLD antibodies immobilized on protein A resin(PLD antibody complex) were incubated with purified recombinant PLD2 (3μg) for 2 hours. After a brief centrifugation, the immune complexes werewashed three times with radioimmune precipitation buffer (50 mMTris/HCl, pH 8.5, 0.1% SDS, 150 mM NaCl, 1% Triton X-100, and 1%deoxycholate). The prepared brain extract (3 mg of protein) was thenincubated with the complexes for 2 hours at 4° C. Finally, theco-precipitated proteins were washed again three times with radioimmuneprecipitation buffer, loaded onto a gel, and visualized by CoomassieBrilliant Blue staining. The result was shown in FIG. 1, which showsanti-PLD antibody complexes in the absence (−) or presence (+) ofrecombinant PLD2 incubated with homogenation buffer (MOCK) or rat brainextract (EXT). As indicated by an arrow in FIG. 1, a 43-kDa protein isdetected as a PLD2-binding protein.

(2) Identification of the 43-kDa Protein

Identification of the 43-kDa protein obtained in the above was performedusing peptide mass fingerprinting by matrix-assisted laser desorptionionization/time-of-flight mass spectrometry, according to J. B. Park et.al., J. Biol. Chem. 275, 21295-21301, 2000. The fraction containing the43-kDa protein (p43) after co-immunoprecipitation from rat brain extractwas separated by 8% SDS-PAGE, and the band corresponding to p43 wasexcised and digested with trypsin (Roche Molecular Biochemicals) for 6hours at 37° C. The masses of the tryptic peptides obtained weredetermined with a Voyager DE time-of-flight mass spectrometer(Perceptive Biosystems, Inc., Framingham, Mass.) in the Korea BasicScience Institute (Busan, Korea). The masses obtained, marked as P1-P7,were compared with protein in the Swiss-Protein database using theMS-Fit peptide mass search program. As shown in Table 1, the peptideexhibited molecular masses that were almost identical to the calculatedmasses of the corresponding theoretically predicted tryptic peptides ofβ-actin. TABLE 1 M + H⁺ Observed Calculated^(b) Peptide Sequence^(a) DaP1 LDLAGR (178-183) 644.36 644.37 P2 ILAPPER (329-335) 795.49 795.47 P3GYSFTTTAER (197-206) 1132.54 1132.52 P4 HQGVMVGMGQK (40-50) 1187.571187.56 P5 QEYDESGPSIVHR (360-372) 1516.72 1516.70 P6 SYELPDGQVITIGNER(239-254) 1790.90 1790.89 P7 VAPEEHPVLLTEAPLNPK 1954.03 1954.06 (96-113)^(a)The matched peptides cover 21% (81 of 375 amino acids) of theproteins^(b)Monoisotopic mass

To substantiate the identity of this protein further, the presence ofactin in the PLD2 precipitate was confirmed using a monoclonal antibodyto actin. As shown in FIG. 2, actin was strongly detected in the PLD2precipitate but not in a control immune complex. On the basis of theseresults, it is concluded that the 43-kDa protein in the PLD2 precipitatefrom the rat brain extract is β-actin.

EXAMPLE 2 Interaction between PLD2 and Aldolase

(1) 40 kDa Protein from Rat Brain was Detected as a PLD2-Direct BinderUsing Blot Overlay Assay.

All preparations were performed at 4° C. or on ice. Adult rat brains(total 30 g) were homogenized using a polytron homogenizer inhomogenation buffer containing 20 mM Tris, pH 7.6, 1 mM MgCl₂, 1 mMPMSF, and 0.1 mM DTT. The homogenate was centrifuged at 100,000 g for 1h and the resulting supernatant (the cytosolic fraction) was collected.The cytosolic fraction (900 mg) was loaded to a Q-Sepharose anionexchange column (13 cm×3 cm) pre-equilibrated with buffer A (20 mM Tris,pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM MgCl₂, and 0.1 mM DTT). Unboundproteins (flow-through fractions) were collected, and NaCl was addedthereto to 2 M. After centrifugation (50,000g, 20 min), the resultingsupernatant was loaded onto a Phenyl Sepharose column (70 cm×2 cm).Proteins were eluted at a flow rate of 2 mL/min by applying a decreasinggradient of NaCl (from 2 M to 0 M) over a period of 60 min. Fractionswere collected and tested by blot overlay assay with purified PLD2. Peakfractions were pooled, and diluted with buffer A to adjust the saltconcentration to 50 mM NaCl, and then loaded onto a Hi-Trap heparincolumn (1 mL, Pharmacia) pre-equilibrated with buffer A containing 50 mMNaCl. Bound proteins were then eluted at a flow rate of 0.5 mL/min usinga linear gradient of 0.05-1 M NaCl over 30 min. Fractions were collectedand tested by blot overlay assay. Fractions containing PLD2-interactingproteins were pooled and continuously loaded onto a Bio Gel HT (1 mL,Bio-Rad) pre-equilibrated with buffer B (20 mM Tris, pH 7.6, 50 mM NaCl,0.1 mM DTT). Bound proteins were eluted at a flow rate of 0.3 mL/min byapplying an increasing gradient of 0-0.25 M KH₂PO₄. Then, fractions werecollected and tested using the blot overlay assay with purified PLD2.PLD2 overlay assay was performed as previously described in J. B. Parket. al., J. Biol. Chem. 275, 21295-21301, 2000. In brief, rat braincytosolic proteins were separated by SDS-PAGE and transferred tonitrocellulose membranes. The blots were preincubated overnight with PLDassay buffer (50 mM HEPES, pH 7.3, 3 mM EGTA, 3 mM CaCl₂, 3 mM MgCl₂, 80mM KC1) containing 0.1 mM DTT and 5% (w/v) skim milk at room temperatureand then incubated with the same buffer containing 1 μg/mL of purifiedPLD2 for 3 h at room temperature. The membranes were washed severaltimes with Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 150 mM NaCl)containing 0.05% Tween 20 and reacted with polyclonal antibodiesdirected against PLD for 3 h. After the washings, the membranes weresubsequently incubated with horseradish peroxidase-conjugated goatanti-rabbit IgG antibodies for 1 h and developed using an enhancedchemiluminescence kit as described by the manufacturer. The techniqueused has been previously described in J. B. Park et. al., J Biol. Chem.275, 21295-21301, 2000. In brief, the fraction containing the 40 kDaprotein (p40) obtained by hydroxyapatite column chromatography wasseparated by 10% SDS-PAGE, and the band corresponding to p40 was excisedand digested with trypsin (Roche Molecular Biochemicals) for 6 h at 37°C. FIG. 3A shows the isolation of PLD2-binding protein (p40) by theabove blot overlay assay.

The masses of the tryptic peptides so obtained were determined using aBruker REFLEX reflector time-of-flight mass spectrometer(Bruker-Franzen, Bremen, Germany). Delayed ion extraction resulted inpeptide masses in 50 ppm mass accuracy or higher on average. Using theamino acid sequences and the mass numbers of the tryptic peptides ofp40, the Swiss-Protein database was searched for a protein match. FIG.3B shows the matrix-assisted laser desorption ionization mass spectrumof the digested peptides of p40. The masses obtained were compared withprotein in the Swiss-Protein database using the MS-Fit peptide masssearch program. The peptide exhibited molecular masses that were almostidentical to the calculated masses of the corresponding theoreticallypredicted trytic peptides of aldolase.

(2) Association between PLD2 and Aldolase.

In vitro binding between all the GST fusion proteins and aldolase wasperformed in PLD assay buffer (50 mM HEPES/NaOH, pH 7.3, 3 mM EGTA, 3 mMCaCl₂, 3 mM MgCl₂, 80 mM KCl) containing 1% Triton X-1 00 for 1 h at 4°C. After a brief centrifugation, the precipitated complexes were washed3 times with the same buffer before being loaded onto a polyacrylamidegel. COS-7 cells were transfected in combination with pCDNA 3.1 vectorharboring human PLD2 and pCMV vector containing aldolase A. The culturedcells were harvested and lysed with PLD assay buffer containing 1%Triton X-100, 1% cholate, and 1 mM PMSF. After a brief sonication, thelysates-were-centrifuged at 100,000 g for 30 min and the cell extracts(1 mg) were incubated respectively with anti-PLD or anit-flagantibody-conjugated protein A Sepharose overnight. After a briefcentrifugation, the co-immunoprecipitated complexes were washed 3 timeswith the same buffer before being loaded onto a gel. As shown in FIG. 4,aldolase strongly interacts with PLD2.

EXAMPLE 3 Interaction between PLD2 and CRMP

(1) Identification of p62 as CRMP-2.

Purified PLD₂-interacting protein from the hydroxylapatite column inExample 2 was digested for 2 h at 37° C. with V8 protease obtained fromStaphylococcus aureus and then subjected to 15% SDS-PAGE to separate thecleaved peptides. After transferring the peptides to a polyvinylidenedifluoride membrane, they were stained with Coomassie Brilliant Blue,rinsed several times with 30% methanol, excised, and subjected to Edmandegradation. The candidate protein was identified by sequencing (ABI473Sequencer) at the Institute of Basic Science (Busan, Korea) and bycomparing the results obtained from the Swiss-Protein database using theBlastP algorithm. The masses obtained were compared with protein in theSwiss-Protein database using the MS-Fit peptide mass search program. Thepeptide exhibited molecular masses that were almost identical to thecalculated masses of the corresponding theoretically predicted trypticpeptides of CRMP-2 (see FIG. 5).

(2) Direct Interaction of CRMP-2 with PLD2 in vitro.

Affinity-purified anti-PLD2 antibodies immobilized with protein A beadswere first incubated with purified PLD2 for 2 h at4° C. After a briefcentrifugation, the precipitates were reincubated with the indicatedamounts of purified CRMP-2 for 15 min at 37° C. in PLD assay buffercontaining 1% Triton X-100. Binding site mapping between PLD2 and CRMP-2was performed by incubating the indicated amounts of glutathioneS-transferase (GST) fusion proteins with purified PLD2 or rat CRMP-2,respectively, under the same buffer conditions for 15 min at 37° C.After a brief centrifugation, the pellets were washed three times withthe same buffer before being loaded onto a polyacrylamide gel. As shownin FIG. 6, CRMP-2 strongly interacts with PLD2.

EXAMPLE 4 Interaction between PLD2 and PLC-γ1

Recombinant rat PLC-β1, PLC-γ1, and human PLD2 were expressed in Sf9cells and purified. Cells were lysed with IP buffer (20 mM Tris/HCl, pH8.0, 150 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 1% cholicacid) by sonication. Cell lysates were centrifuged at 100,000×g at 4° C.for 30 min, and the supernatants were incubated with an antibodyimmobilized to Protein A-Sepharose beads. In vitro binding was performedin 300 μl of binding buffer (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mMMgCl₂, 1 mM EGTA, 0.1% Triton X-100) at 4° C. for 3 h. Proteins weredenatured by boiling for 5 min at 95° C. in a Laemmli sample buffer,separated by SDS-PAGE, and immunoblot analysis was performed asdescribed previously. As shown in FIG. 7, PLC-γ1 strongly interacts withPLD2.

EXAMPLE 5 Interaction between PLD2 and GAPDH

(1) H₂O₂-Dependent Association of p35 to PLD2 in PC12 Cells.

PLD2-inducible PC12 cells were subcultured in the presence or absence of0.5 μg/mL tetracycline for 24 h. After additional 24 h of serumdeprivation, cells were treated with H₂O₂ or KA and washed with ice-coldbuffer A (50 mM HEPES/NaOH, pH 7.5, 80 mM KCl, 2.5 mM MgCl₂, 3 mM EGTA)containing protease inhibitors twice and harvested by scraping.Inhibitors were pre-incubated when required and included duringincubation periods. Cells were lysed with buffer A containing 1%n-octyl-β-D-glucopyranoside and protease inhibitors by brief sonication.Soluble extracts were obtained by centrifugation for 1 h, 100,000×g at4° C. Resulting supernatants were incubated with anti-PLD antibody (5μg) coupled to Protein A Sepharose bead at 4° C. After 5 h ofincubation, resulting pellets were washed four times with buffer Acontaining 1% n-octyl-β-D-glucopyranoside before being separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis and silverstaining or immunoblotting. As indicated by an arrow in FIG. 8, a 35-kDaprotein is detected as a PLD2-binding protein.

(2) Identification of p35 as Glyceraldehyde 3-Phosphate Dehydrogenase.

Protein band excised from gels were subjected to in-gel digestion withtrypsin following the method reported after intensive destaining with40% methanol (Jensen et aL 1999). Spectra were obtained using a BrukerReflex III matrix assisted laser desorption/ionization time-of-flightmass spectrometer. Tryptic peptides were desalted and concentrated withhomemade nano-scale reverse-phase columns using GeLoader tip asdescribed previously (Gobom et al. 1999). Peptide mixtures were loadeddirectly to MALDI probes using a 20 mg/mL alpha-cyano-4-hydroxycinnamicacid (Sigma) as a matrix. Spectra were calibrated internally usingtrypsin autodigestion products (m/z 842.51 and 2211.10). The searchprogram ProFound (http://www.proteometrics.com) was used for searches inthe database NCBlnr (Zhang and Chait 2000). The matrix-assisted laserdesorption ionization mass spectrum of the digested peptide of p35exhibits molecular masses almost identical to the calculated masses ofthe corresponding theoretically predicted tryptic peptides of that wasglyceraldehyde 3-phosphate dehydrogenase.

EXAMPLE 6 Interaction between PLD2 and Akt1

COS7 cells in serum growing states were lysed with Buffer B containing1% cholic acid, 1% Triton X-100, protease inhibitor and phosphataseinhibitor cocktail. After extensive sonication, lysates were clarifiedby centrifugation at 15,000 rpm. 1.5 mg of lysates were incubated witheither anti-PLD antibody or control antibody, precoupled to Protein Aagarose bead, for over 4 hrs at 4° C. After being washed with Buffer Bfive times, the complexes were subjected to immunoblot analysis and theexistence of Akt was revealed by specific anti-Akt monoclonal antibody.COS 7 cells were cotransfected with either PLD1 or PLD2 with wild-typeAkt as indicated. Cells were lysed and sonicated in Buffer B containing1% cholic acid and 1% Triton X-100. After immunoprecipitation withanti-PLD antibody, followed by washing three times, the amount ofproteins in precipitates was revealed by indicated antibody. Resultswere reproduced in two dependent experiments. Akt (100 ng) purified frombaculo virus-infected Sf9 cells was incubated in the absence (−) orpresence (+) of purified PLD1 for 30min at 4 ° C. Akt/PLD1 complex wasisolated by anti-PLD antibody precoupled onto Protein A agarose bead foranother 30 min at 4° C. After being washed with buffer A containing 0.5%Triton X-100 and 0.3% BSA three times, the complexes were subjected toimmunoblot analysis using anti-PLD antibody and anti-myc antibody.Prebound Akt onto anti-myc antibody was incubated with either purifiedPLD1 or PLD2 for 1 hr at 4° C. After washing with buffer A containing0.5% Triton X-100 and 0.3% BSA three times, the complexes were subjectedto immunoblot analysis and the amount of coprecipitated Akt was revealedby anti-myc antibody. All the results shown are representative of twoindependent experiments. As shown in FIG. 9, PLD1 forms peptidecomplexes with Akt strongly and specifically both in vitro and in vivo.

EXAMPLE 7 Interaction between PLD2 and Glucose Transporter 4

(1) Association with PLD1 and GLUT4.

COS-7 cells were transfected in combination with pcDNA 3.1 vectorharboring PLD1 and HA-GLUT4. The cultured cells were harvested and lysedwith PLD assay buffer containing 1% Triton X-100, 1% cholate, and 1 mMPMSF. After a brief sonication, the lysates were centrifuged at100,000×g for 30 min and the cell extracts (2 mg) were incubated withanti-PLD body-conjugated protein A Sepharose for 4 hrs. After a briefcentrifugation, the co-immunoprecipitated complexes were washed threetimes with the same buffer before being loaded onto a gel. FIG. 10 showsassociation with PLD1 and GLUT4.

(2) Direct Binding of Purified PLD1 to the Cytoplasmic Central Loop ofGLUT4.

All experiments were performed at 4° C. on ice. In vitro binding betweenthe GST-fusion proteins corresponding to N, C-termius and central loopof GLUT and PLD1 and between GST-fragments of PLD1 and MBP-fusioncentral loop of GLUT4 were performed in the PLD assay buffer (50 mMHepes/NaOH, pH 7.4, 3 mM EGTA, 3 mM CaCl₂.3 mM MgCl₂, 80 mM KCl)containing 1% Triton X-100 and 5% glycerol for 1.5 hrs at 4° C. After abrief centrifugation, the precipitated complexes were washed three timeswith the same buffer before being loaded onto a polyacrylamide gel.Affinity-purified anti-PLD1 antibody immobilized with protein A beadswas first incubated with purified PLD1 for 2 h at 4° C. After washing,the precipitates were reincubated with the indicated amounts ofsolubilized GST-fusion central loop of GLUT4 for 1 hr at 4° C in PLDassay buffer containing 1% Triton X-100 and 5% glycerol. After a briefcentrifugation, the precipitated complexes were washed three times withthe same buffer before being loaded onto a polyacrylamide gel. FIG. 11shows a direct binding of purified PLD1 to the cytoplasmic central loopof GLUT4.

EXAMPLE 8 Interaction between PLD2 and Heat Shock 70

(1) Identification of p70 as HSP70

PLD2-inducible PC12 cells were subcultured in the presence or absence of0.5 g/ml tetracycline for 24 h. Cells were lysed in lysis buffer (50 mMHEPES, pH 7.3, 3 mM EGTA, 2 mM CaCl₂, 3 mM MgCl₂, 80 mM KC1 and 1 mMdithiothreitol, 1% Triton X-100, and 1% sodium cholate) containingprotease inhibitors. After centrifugation (12,000×g for 15 min), equalamount of soluble extract was incubated with anti-PLD antibodyimmobilized on protein A Sepharose. After 4 h of incubation, immunecomplexes were washed four times with lysis buffer before separated bySDS-PAGE. As shown in FIGS. 12 and 13, HSP70 is detected as aPLD2-binding protein.

(2) Direct Interaction of HSP70 and PLD2

Protein peptide fingerprinting analysis was performed as described (17).In brief, the candidate band was excised from the gel and digested withtrypsin. A 1 μl aliquot of the total digest (total volume 30 μl) wasused for peptide mass fingerprinting. The masses of the tryptic peptideswere measured with a Bruker Reflex III mass spectrometer.Matrix-assisted laser desorption/ionization (MALDI) was performed withα-cyano-4-hydroxycinnamic acid as the matrix. Trypsin autolysis productswere used for internal calibration. Delayed ion extraction resulted inpeptide masses with better than 50 ppm mass accuracy on average.Comparison of the mass values against the Swiss-Protein database wasperformed by Profound. FIG. 14 shows a direct interaction of HSP70 andPLD2.

EXAMPLE 9 Interaction between PLD2 and Dynamin

(1) Identification of Dynamin as PLD-Interacting Protein from Rat Brain

Rat brains were lysed with buffer A (20 mM Tris/HCl, pH 8.0, 150 mMNaCl, 1 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 1% cholic acid) bysonication. The lysates were centrifuged at 100,000×g at 4° C. for 30min, and the supernatants were incubated with anti-PLD antibodyimmobilized to Protein A-Sepharose beads. The resulting immune complexeswere pelleted down and washed three times with buffer A and subjected toSDS-PAGE followed by silver staining. After stained with silverstaining, the candidate band was excised from the gel and digested withtrypsin as described. A 1 μl aliquot of the total digest (total volume30 μl) was used for peptide mass fingerprinting. The masses of thetryptic peptides were measured with a Bruker Reflex III massspectrometer. Matrix-assisted laser desorption/ionization (MALDI) wasperformed with α-cyano-4-hydroxycinnamic acid as the matrix.Matrix-assisted laser desorption/ionization (MALDI) was performed withα-cyano-4-hydroxycinnamic acid as the matrix. Trypsin autolysis productswere used for internal calibration. Delayed ion extraction resulted inpeptide masses with better than 50 ppm mass accuracy on average.Comparison of the mass values against the Swiss-Protein database wasperformed using Peptide Search. As shown in FIGS. 15A and 15B, dynaminis detected as a PLD2-binding protein.

(2) Dynamin Interacts with PLD2 in a GTP-Dependent Manner

In vitro binding of purified dynamin, DYN-K44A, various GST fusedproteins of dynamin PLD2, and PLD2 domains was performed with 300 ml ofPH buffer containing 1% Triton X-100, 1% cholic acid at 4° C. for 3 h inthe presence of 500 mM GTP□S or GDP□S. After washing three times withthe binding buffer, the samples were subjected to SDS-PAGE followed byimmunoblot analysis. FIG. 16 shows a direct interaction of dynamin andPLD2 in a GTP-dependent manner.

EXAMPLE 10 Interaction between PLD2 and Munc 18

(1) Munc-18-1 was Identified as a PLD-Interacting Protein from Rat BrainMembrane.

Rat brains (3 g) were lysed with buffer A (20 mM Tris/HCl, pH 8.0, 150mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 1% cholic acid) byhomogenization. The lysates were centrifuged at 100,000×g at 4° C. for30 min, and the supernatants were incubated with anti-PLD antibodyimmobilized to Protein A-Sepharose beads. As shown in FIG. 17, munc-18-1is identified as a PLD-interacting protein.

(2) Munc-18-1 Direct Interacts with PLD2 in a Dose-Dependent Manner.

Cells were lysed with PLD assay buffer (50 mM HEPES/NaOH, pH 7.3, 3 mMEGTA, 3 mM CaCl₂, 3 mM MgCl₂, and 80 mM KC1) containing 1% Triton X-100,1% cholic acid, 1 mM phenylmethylsulfonyl fluoride. After briefsonication, the cell lysates were centrifuged at 100,000×g for 30 min at4° C. The supernatants (1 mg of protein) were incubated -with anti-PLDantibody-immobilized on protein A resin for 6 h at 4° C. Proteins weredenatured by boiling for 5 min at 95° C. in a Laemmli sample buffer,separated by SDS-PAGE, and immunoblot analysis was performed asdescribed previously. In vitro binding was performed in 300 μl of PLDassay buffer (50 mM HEPES/NaOH, pH 7.3, 3 mM EGTA, 3 mM CaCl₂, 3 mMMgCl₂, and 80 mM KC1) containing 0.1% Triton X-100 and 0.1% cholic acidat 4° C. for 2 h. FIG. 18 shows a direct interaction of munc-18-1 andPLD2 in a dose-dependent manner.

EXAMPLE 11 Interaction between PLD2 and Tubulin

(1) p55 Protein was Co-Immunoprecipitatd with PLD2 at PLD2 Activity TurnOff Time.

The cultured cells were harvested and lysed with PLD assay buffer (50 mMHepes/NaOH, pH 7.3, 3 mM EGTA, 3 mM CaCl₂.3 mM MgCl₂, 80 mM KCl)containing 0.5% TX-100, and 1% Cholic acid, 1 mM PMSF, 1 μg/mlleupeptin, and 5 μg/ml aprotinin. After a brief sonication, the lysateswere centrifuged at 100,000×g for 1 hr, and the cell extracts wereincubated respectively with immobilized anti-PLD antibody for overnight.After a brief centrifugation, the co-immunoprecipitated complexes werewashed five times with the same buffer before being loaded onto a gel.In vivo PLD activity was determined as described previously. In brief,vector or human PLD2-transfected COS-7 cells were cultured for 48 h. Thecells were loaded with [³H]myristic acid (10 μCi/ml) for 4 h and thenwashed twice with DMEM. And COS-7 cells were pre-incubated withNocodazole and Taxol for 20 min. The loaded cells were incubated with0.4% butanol for 5 min and, scraped into 0.8 ml of methanol and 1 M NaCl(1:1), and mixed with 0.4 ml of chloroform. The organic phases weredried, and the lipids were separated by thin-layer chromatography onsilica-gel plates. The PLD activity of PLD2-overexpressing PC12 cellswas determined using the same procedures. The amount of[³H]phosphatidylbutanol formed was expressed as a percentage of thetotal ³H-lipid to account for cell labeling efficiency differences. Asshown in FIG. 19, a 55-kDa protein is detected as a PLD2-bindingprotein.

(2) The 55-kDa Protein Precipitated with PLD2 in COS-7 Cells wasIdentified as α-, β-tubulin.

The technique used in this example followed as described previously. Inbrief, the fraction containing 55-kDa protein (p55) afterco-immunoprecipitation from COS-7 cells was separated by SDS-PAGE, andthe band corresponding to p55 was excised and digested with trypsin(Roche Molecular Biochemicals) for 6 h at 37° C. Delayed ion extractionresulted in peptide masses with better than 50 ppm mass accuracy onaverage. Using the amino acid sequences and the mass numbers of thetryptic peptides of p50, the Swiss-Protein database was searched for aprotein match. In vitro binding of all the GST fusion PLD2 fragmentproteins and immune complex with tubulin was performed in the PLD assaybuffer (50 mM Hepes/NaOH, pH 7.3, 3 mM EGTA, 3 mM CaCl₂.3 mM MgCl₂, 80mM KCl) containing 0.1% Triton X-100, 1 mM PMSF, 1 μg/ml leupeptin, and5 μg/ml aprotinin for 20 min at 37° C. After a brief centrifugation, theprecipitated complexes were washed five times with the same bufferbefore being loaded onto a polyacrylamide gel. FIG. 20 shows that the55-kDa protein precipitated with PLD2 in COS-7 cells is α- andβ-tubulin.

EXAMPLE 12 Interaction between PLD2 and nNOS

(1) Neuronal Nitric Oxide Synthase (nNOS) was Identified as aPLD-Interacting Protein in Rat Brain.

The same procedures as in Example 9 (1) were repeated to identify nNOSas a PLD-interacting protein in rat brain (see FIG. 21).

(2) nNOS More Strongly Interacted with PLD2 than PLD1 in COS-7 Cells

The same procedures as in Example 7 (1) were repeated using pcDNA 3.1vector harboring PLD1 and pCMV-Flag-2 vector encoding nNOS or pcDNA 3.1vector harboring PLD2 and pCMV-Flag-2 vector encoding nNOS. FIG. 22shows a strong interaction between nNOS and PLD2.

EXAMPLE 13 Interaction between PLD2 and Integrin Beta 3

(1) PLD Directly Interacts with Integrin Beta 3, 5 Cytosolic Tail.

The same procedures as in Example 7 (2) were repeated to identify adirect interaction between PLD2 and integrin beta 3, 5 cytosolic tail(see FIG. 23).

(2) PLD Interacts with Integrin Beta3 in COS 7 Cells.

The same procedures as in Example 7 (1) were repeated using pcDNA 3.1vector harboring PLD1 and pCMV-Flag-2 vector encoding integrin beta3 orpcDNA 3.1 vector harboring PLD2 and pCMV-Flag-2 vector encoding integrinbeta3. FIG. 24 shows PLD directly interacts with integrin beta 3 in COS7cells.

EXAMPLE 14 Interaction between PLD1 & 2 and mTOR

Binding mapping analysis and translation assay were performed with PLD1& 2, using the procedure as in Example 7 (1). FIG. 25 shows aninteraction between PLD 1 & 2 and mTOR.

EXAMPLE 15 Interaction between PLD2 and PIP3

A protein-lipid overlay assay was performed using GST fusion proteins aspreviously described. Lipids were dissolved in chloroform: methanol:water (1: 2: 0.8 volume ratio) and 1 μl aliquotes containing 0.2, 0.5,and 1 nmol phosphoinositides were spotted onto Hybond-C extra-membranes(Amersham Pharmacia Biotech). After drying at room temperature for 1 h,the membranes were blocked in 3% fat-free BSA (Sigma-Aldrich) in TNE (50mM Tris-HCl, pH 7.5,150 mM NaCl, 1 mM EDTA) with 0.1% Tween-20 at 4° C.overnight. Membranes were then incubated with 1 μg/ml of purifiedGST-PLD1 wild type, R179K, or R179A mutant respectively at roomtemperature for 1 h, washed five times with TNE with 0.1% Tween-20, andincubated with anti-GST antibody. After 1 h washing, the membranes werefurther incubated with horseradish peroxidase-conjugated goat anti-mouseantibody for 1 h at room temperature and then washed again for 1 h. Thesignals were detected by enhanced chemiluminescence kit. Phospholipidsvesicles composed of 53 μM phosphatidylcholine (PE), 3.3 μMphosphatidylcholine (PC), and 4.6 μM phosphoinositides (Ptdlns) wereincubated with 10 ng of purified GST-PLD1 PX domains in 150 μl buffercontaining 50 mM Hepes-NaOH, pH 7.5, 3 mM MgCl₂, 2 mM CaCl₂, 3 mM EGTA,and 80 mM KCl as previously described (Kim et al., 1998). Afterincubation at 37° C. for 15 min, the reaction mixtures were centrifugedat 300,000×g for 30 min in a TL-100 ultracentrifuge (Beckman). Theresulting supernatants and pellets were subjected to 8% SDS-PAGE andimmunoblotted using anti-GST antibody. FIGS. 26A, 26B, and 26C showshows PLD1 PX domain has high affinity with phosphoinositide-3-phosphate(PIP3).

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. An isolated peptide complex comprising: a first peptide selected fromthe group consisting of: (a1) phospholipase D (PLD), (a2) a PLD variant,(a3) a PLD fragment, and (a4) a fusion peptide containing (a1), (a2), or(a3); and a second peptide selected from the group consisting of (b1)actin, glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycin (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), or dopamine transporter(DAT), (b2) a variant of (b1), (b3) a fragment of (b1), and (b4) afusion peptide containing (b1), (b2), or (b3).
 2. The isolated peptidecomplex of claim 1, wherein the first peptide is PLD and the secondpeptide is selected from the group consisting of actin,glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1, glucosetransporter 4 (GLUT4), mammalian target of rapamycine (mTOR), heat shockprotein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxide synthase(nNOS), integrin beta 3, guanine nucleotide exchange factor—H1 (GEF-H1),V-ATPase, phosphoinositide-3-phosphate (PIP3), dopamine transporter(DAT).
 3. The isolated peptide complex of claim 1, wherein the firstpeptide is the fusion peptide containing PLD, a PLD variant or a PLDfragment.
 4. The isolated peptide complex of claim 1, wherein the secondpeptide is the fusion peptide containing one or more peptide selectedfrom the group consisting of actin, glyceraldehyde-3-phosphatedehydrolase (GAPDH), Akt1, glucose transporter 4 (GLUT4), mammaliantarget of rapamycine (mTOR), heat shock protein 70 (hsp70), dynamin,munc 18, tubulin, n-nitric oxide synthase (nNOS), integrin beta 3,guanine nucleotide exchange factor—H1 (GEF-H1), V-ATPase,phosphoinositide-3-phosphate (PIP3), dopamine transporter (DAT), avariant thereof, and a fragment thereof.
 5. The isolated peptide complexof claim 1, wherein the first peptide is linked to the second peptide bya covalent bond.
 6. A screening method for modulators of the peptidecomplex according to claim 1, which comprises: providing the isolatedpeptide complex; contacting the isolated peptide complex with a testcompound; and detecting an interaction between the test compound and theisolated peptide complex and/or an interaction change between the firstpeptide and the second peptide.
 7. A screening method for modulators ofan interaction between a first peptide selected from the groupconsisting of (a1) phospholipase D (PLD), (a2) a PLD variant, (a3) a PLDfragment, and (a4) a fusion peptide containing (a1), (a2), or (a3); anda second peptide selected from the group consisting of (b1) actin,aldolase, collapsin response mediator molecule-2 (CRMP-2), phospholipaseC-γ1 (PLC-γ1), glyceraldehyde-3-phosphate dehydrolase (GAPDH), Akt1,glucose transporter 4 (GLUT4), mammalian target of rapamycin (mTOR),heat shock protein 70 (hsp70), dynamin, munc 18, tubulin, n-nitric oxidesynthase (nNOS), integrin beta 3, guanine nucleotide exchangefactor—H1(GEF-H1), V-ATPase, phosphoinositide-3-phosphate (PIP3), ordopamine transporter (DAT), (b2) a variant of (b1), (b3) a fragment of(b1), and (b4) a fusion peptide containing (b1), (b2), or (b3), whichcomprises: contacting the first peptide with the second peptide inpresence of a test compound; and detecting an interaction between thefirst peptide and the second peptide.
 8. The screening method of claim,wherein at least one of the first and second peptides is a fusionpeptide having a detectable tag.
 9. The screening method claim, whereinthe contacting step is conducted in a substantially cell freeenvironment.
 10. The screening method claim, wherein the interaction orinteraction change between the first peptide and the second peptide isdetermined in a host cell.
 11. The screening method claim, wherein thedetecting comprises measuring the amount of the peptide complex formedwith the first and second peptides.
 12. The screening method claim,further comprising generating a data set defining one or more selectedtest compounds.
 13. The screening method claim 12, wherein the data setis in a transmittable form.